Invasive and Noninvasive Approaches in Prenatal Diagnosis of Thalassemias

Thalassemia is a significant health problem worldwide. There are two main classifications, α- and β-thalassemias, which are usually caused by the defective synthesis of the α-globin, and which are commonly caused by different mutations of the β-globin chain. Different hemoglobin mutations have been identified to date. Thalassemias can result in profound anemia from early life and, if not treated with regular blood transfusions, can lead to death in the first year. Prenatal diagnosis of thalassemia is the essential part of preventive medicine and is currently dependent on the use of invasive diagnostic tests within the first 2 months of pregnancy. These diagnostic techniques carry a small but significant risk of fetal loss up to 1%. Molecular diagnostic methods have been developed for genotyping thalassemias based on PCR techniques and high-throughput technologies. Noninvasive tests using cell-free DNA (cfDNA) from a maternal blood sample is also an alternative method, thus eliminating the risk of miscarriage. This chapter summarizes the current invasive approaches and the noninvasive methods using cell-free fetal DNA as new molecular diagnostic methods for genotypic diagnosis of thalassemia in clinical practice. Prevention strategies that encompass carrier screening, genetic counseling, and prenatal diagnosis are discussed

Prenatal Diagnosis of β-Thalassemias and Hemoglobinopathies.

Prenatal diagnosis of β-thalassemia was accomplished for the first time in the 1970s by globin chain synthesis analysis on fetal blood obtained by placental aspiration at 18–22 weeks gestation. Since then, the molecular definition of the β-globin gene pathology, the development of procedures of DNA analysis, and the introduction of chorionic villous sampling have dramatically improved prenatal diagnosis of this disease and of related disorders. Much information is now available about the molecular mechanisms of the diseases and the molecular testing is widespread. As prenatal diagnosis has to provide an accurate, safe and early result, an efficient screening of the population and a rapid molecular characterization of the couple at risk, are necessary prerequisites. In the last decades earlier and less invasive approaches for prenatal diagnosis were developed. A overview of the most promising procedure will be done. Moreover, in order to reduce the choice of interrupting the pregnancy in case of affected fetus, Preimplantation or Preconceptional Genetic Diagnosis (PGD) has been setting up for several diseases including thalassemia

EMQN best practice guidelines for molecular and haematology methods for carrier identification and prenatal diagnosis of the haemoglobinopathies

Haemoglobinopathies constitute the commonest recessive monogenic disorders worldwide, and the treatment of affected individuals presents a substantial global disease burden. Carrier identification and prenatal diagnosis represent valuable procedures that identify couples at risk for having affected children, so that they can be offered options to have healthy offspring. Molecular diagnosis facilitates prenatal diagnosis and definitive diagnosis of carriers and patients (especially ‘atypical’ cases who often have complex genotype interactions). However, the haemoglobin disorders are unique among all genetic diseases in that identification of carriers is preferable by haematological (biochemical) tests rather than DNA analysis. These Best Practice guidelines offer an overview of recommended strategies and methods for carrier identification and prenatal diagnosis of haemoglobinopathies, and emphasize the importance of appropriately applying and interpreting haematological tests in supporting the optimum application and evaluation of globin gene DNA analysis

An Early Diagnosis of Thalassemia: A Boon to a Healthy Society

The β-thalassemia is a hereditary blood disorders, characterized by reduced or absent synthesis of the hemoglobin beta chain that cause microcytic hypochromic anemia. An early diagnosis, economical test, awareness programs and prenatal screening will be a milestone for the eradication of this genetic disorder and to reduce burden of the health sector of a country subsequently the economics. Initially, the diagnosis of β-thalassemia depends on the hematological tests with red cell indices that disclosed the microcytic hypochromic anemia. Hemoglobin analysis shows the abnormal peripheral blood smear with nucleated red blood cells, and reduced amounts of hemoglobin A (HbA). In severe anemia, the hemoglobin analysis by HPLC reveals decreased quantities of HbA and increased the level of hemoglobin F (HbF). The decrease level of MCV and MCH are also associated with β-thalassemia. There are various different molecular techniques such as ARMS PCR, allele-specific PCR, Gap PCR, denaturing gradient gel electrophoresis, reverse dot blotting, DGGE, SSCP, HRM, MLPA, sequencing technology and microarray available to identify the globin chain gene mutations. These molecular techniques can be clustered for detection by mutation types and alteration in gene sequences

New Perspectives in Prenatal Diagnosis of Sickle Cell Anemia

Hemoglobin disorders such as thalassemias and sickle cell anemias can be avoided by detecting carriers, ensuring genetic counseling and prenatal diagnosis. Nowadays Chorionic villus sampling (CVS amniocentesis, and cordocentesis are still the most widely used invasive sampling methods for prenatal diagnosis of the fetus. These traditional methods are associated with a risk of fetal loss. The revelation of cell-free fetal DNA (cffDNA) in maternal plasma and serum provides the opportunity of noninvasive prenatal diagnosis (NIPD). Different encouraging clinical applications have arose such as noninvasive identification of fetal sexing, fetal Rhesus D, and the determination of the paternal alleles in maternal plasma. The determination of the presence or absence of paternally inherited alleles in maternal plasma of sickle cell disease (SCD) and β-thalassemia would allow the diagnosis of autosomal dominant diseases or the exclusion of autosomal recessive diseases of the fetuses, respectively. prenatal diagnosis of genetic diseases. Analysis of cffDNA in maternal plasma for NIPD has the advantage of being safer versus the invasive methods. Different technologies were used since the discovery of cffDNA for NIPD—especially high-resolution melting (HRM) analysis is one of those methods. Genotyping can be done with HRM without using labeled probes and more complex regions can be analyzed with unlabeled hybridization probes. High-resolution melting is a rapid and useful method to detect paternal alleles for the NIPD of SCD and thalassemias when the fetus has a risk for double heterozygote

Newborn Screening and Thalassaemia Syndrome

Haemoglobin variants or haemoglobin disorders are a group of clinical disorders characterised by impairment of synthesis of normal adult haemoglobin, due to genetically determined abnormality in the formation of the globin moiety of the molecule. These disorders fall into two broad groups, that is qualitative (haemoglobinopathies) and quantitative (thalassaemias). In the anthropoids, the most common congenital single-gene disorder is the alteration of the globin genes which account for about 270 million carriers globally. These globin gene alterations cause low/no globin expression (thalassaemia) or abnormal globin protein production (haemoglobinopathy). The clinical manifestation of haemoglobin disorder is the culminated measure of one’s genetic and molecular makeup. Summarily, the study, diagnosis and management of thalassaemia are models of biological principles of human disease. Newborn screening, however, is a system that aims at improving management and/or eradication of genetic disorders from the neonatal stage of life. This chapter will be dealing with the definition and steps involved in newborn screening for thalassaemia

Molecular methods in preimplantation genetic diagnosis with emphasis on the Fragile X syndrome

Preimplantation genetic diagnosis (PGD) is a form of prenatal diagnosis. It is performed on the third day after fertilisation, on embryos of couples at risk of transmitting an inherited disorder to their offspring. In this way, only embryos without the mutation under investigation are transferred in order to initiate a pregnancy. Embryos used for PGD are generated by routine IVF procedures. Fragile X syndrome is the most common form of inherited mental retardation. The causative mutation has been identified as an expansion of a triplet (CGG)n repeat in the 5' untranslated region of the FMR1 gene. The expansion is refractory to PCR due to preferential amplification of the smaller allele in heterozygous cells and the high GC content of the repeat and surrounding sequences. Currently, the only method of diagnosis available for this disease is PCR followed by Southern blotting in order to detect the expanded allele. This, however, is not suitable for preimplantation diagnosis as it is time consuming and requires a lot of DNA. Furthermore, the time at which the amplification occurs in the embryo is not yet determined so that a test relying on detecting the expansion may not be suitable for preimplantation diagnosis. For this reason polymorphic markers linked to the mutation are used to diagnose this disorder in preimplantation embryos. Meanwhile methods for the direct detection of the mutation in single cells have been tested in order to study the timing of the expansion in embryos diagnosed as affected after preimplantation genetic diagnosis. These were applied to spare oocytes and embryos from an IVF cycle. Finally, methods for PGD of sickle cell anaemia have been tested in order to determine the most efficient one. These include restriction enzyme digestion of PCR products, single strand conformation polymorphism (SSCP) followed by staining of the DNA with the silver staining technique, and fluorescent PCR followed by SSCP on an automated fluorescent sequencer (ALF, Pharmacia)

Prevalence of sickle cell gene in Yemen.

To determine the prevalence of the sickle cell gene (HbS) in Yemen and amongst people from different regions of the country living in the capital, Sana'a City, cord blood samples from 1500 consented mothers were collected from hospitals in Sana'a City between July and December 2001. The names and original homes of the parents were recorded. Cationic HPLC analysis was used for screening while isoelectric focusing (IEF) and DNA- PCR were used to confirm haemoglobin S (HbS). Thirty-three samples were found to show Hb FAS giving an overall likely Hb S gene frequency of 0.011. The Hb S gene frequency varied with the part of the country from which the parents came. Amongst people from Taiz and Haja in the west the gene frequency was more than 0.04 but less than 0.004 amongst people from Ibb, adjacent to the governorate of Taiz. Of 66 chromosomes from babies carrying HbS, only 1.5% additionally carried the presence of-158 (C→T) G-gamma globin gene Xmm I site compared with 16.1% of 168 chromosomes from babies without Hb S from the same regions of the sickle cell trait samples identified in this study indicated that the beta S haplotype in not that associated with a milder course found in east Saudi Arabia. In addition to the absence of both Hind III/Ggamma and Hind III/Agamma beta globin polymorphic sites in 26 sickle cell trait samples suggesting the predominant of the African sickle cell haplotype (Benin) in Yemen. The results of this study thus show a higher Hb S gene frequency in the western coastal part of Yemen than in the central mountainous and eastern desert areas. The incidence of affected homozygous births may therefore reach 20/10,000 in the western coastal part of Yemen. A survey to evaluate health care of sickle cell patients was performed using 86 patients attending hospitals in Sanaa City, Yemen. The results showed that the clinical services provided to the sickle cell patients in Yemen were generally very poor. Limited health resources can best be invested in developing a program of (Education, screening and health care initially prioritising those communities residing in the western areas of Yemen with the highest Hb S gene frequency

An approach for the prevention of thalassaemia in Pakistan

The basic aim of this thesis was to identify a suitable approach for prevention of thalassaemia in Pakistan. The overall carrier rate for β-thalassaemia was 5.3%. The rate varied by ethnic group, i.e. Punjabi: 4.5%, Pathan: 5.2%, Sindhi: 1.3%, Baluchi: 9% and Mohajir: 5.2%. The annual birth rate of affected children was estimated at 1.35 per 1000. Screening for α-thalassaemia showed 6% carrier rate for α -3.7 genotype. Approaches for identifying carriers and at risk couples were investigated in pregnant females, but proved to be technically difficult and cost ineffective. By contrast screening in ten index families with haemoglobin disorders identified 21-70% (mean 31%) carriers per family. No carrier was identified in five control families without a history of haemoglobin disorder. Follow-up for one year in the families screened showed a significant effect on the marriage choices. Molecular basis of thalassaemia was investigated in 1240 mutant alleles in all ethnic groups. 19 different mutations were found including one novel allele. The five commonest mutations accounted for 81% of the alleles. The pattern of mutations was significantly different in the ethnic groups studied. 7.5% of patients on blood transfusions had thalassaemia intermedia, and its genetic basis included Xmn-1 +/+ genotype (36%) and mild mutations (31%). Coincidental α-thalassaemia was found or suspected in 38% of cases of thalassaemia intermedia. Prenatal diagnosis of thalassaemia was introduced for the first time in Pakistan and in the two years of the study 158 couples used the test. All couples, except two, already had at least one affected child. They had better education and socio-economic status than the controls and 93% requested termination of pregnancy when the fetus was affected. Over 98% of the diagnoses were done by direct mutation analysis. A multiplex polymerase chain reaction for mutation analysis was developed and significantly reduced the total cost and time required for prenatal diagnosis. Consanguineous marriage and recessive disorders were studied in the nine index and the five control families. In 319 couples studied 46% were consanguineous, 52% were Biradri/Tribe members, and only 6% were completely unrelated. Consanguineous marriage (2nd cousins or closer) had increased from 12% in the 1St generation to 45% in the 2nd generation. Antecedent consanguinity had significant effect on the kinship coefficient of the close as well as distant relatives. There was only a marginal difference in coefficient of inbreeding, calculated in a random population sample by conventional methods (0.0257) and by allele frequencies at the D21S11 locus (0.0272). Morbidity and mortality from genetic causes was significantly higher in the children of consanguineous than the non-consanguineous couples living under similar socio-economic conditions. This pilot study appears to have identified a suitable approach for prevention of thalassaemia in Pakistan